Limitation of photosynthesis and light activation of ribulose, 1,5-bisphosphate carboxylase (RuBPCO) were examined in the 5th leaf of seedlings of red clover (Trifolium pratense L. cv. Renova) for 5 d following an increase in photosynthetic photon flux density (PPFD) from 200 to 550μmol quanta m−2 s−1. Net photosynthesis and its stimulation at 2.0 kPa O2 initial activity of rapidly extracted RuBPCO, standard activity of RuBPCO after incubation of the extracts in the presence of CO2, Mg2+, and inorganic phosphate and contents of soluble protein, starch, soluble sugars, and various photosynthetic metabolites were determined. Photosynthesis decreased and starch content increased. No decrease in photosynthesis was found if, when PPFD was increased, all leaves except the investigated 5th leaf were removed, suggesting that the decrease in photosynthesis was due to accumulated carbohydrates. The stimulation of photosynthesis at 2.0 kPa O2 did not decrease and the ratio of the total foliar steady-state contents of triose phosphate to 3-phosphoglycerate increased suggesting that the decrease in photosynthesis was not due to limiting inorganic phosphate in chloroplasts. Intercellular CO2 partial pressure and RuBP content were not decreased. Nevertheless, the ratio of photosynthesis to initial RuBPCO activity decreased, suggesting that the catalysis per active RuBPCO site was decreased. The increase in PPFD in the growth cabinet and the PPFD at which leaves were preconditioned for 1 h, affected not only initial activity but also the standard activity of RuBPCO. The results suggest that a varying proportion of RuBPCO was bound to membranes and was contained in the insoluble fraction of the extracts. A comparison of photosynthesis with extracted RuBPCO activity suggested that membrane bound RuBPCO did not contribute to photosynthetic CO2 fixation and that the binding and release to and from membranes modulated actual RuBPCO activity in viv
